Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism

Abstract

Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing “molecular bellows” model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“…However, an alternative mode of operation could not be fully ruled out, as recent analysis of MacAB-TolC incorporated into liposomes containing quantum-dots suggested close synchronization of the substrate efflux with ATP-hydrolysis. Furthermore, using the same system did not detect any transfer in the vanadate-treated sample . While a power-stroke associated with ATP-hydrolysis may be an explanation for such behavior, a closer analysis of the MacAB-assembly provides one intriguing alternative.…”

“…Furthermore, using the same system did not detect any transfer in the vanadate-treated sample. 839 While a power-stroke associated with ATP-hydrolysis may be an explanation for such behavior, a closer analysis of the MacAB-assembly provides one intriguing alternative. As mentioned previously, the crown formed of the β-barrel gasket and the lipoyl domain gasket is much-more loosely associated with the transporter than in the case of RND-based assemblies, and it presents a domelike structure, which along with the gating-loops of the lipoyl domain ( Figure 50 and Figure 52 ) creates a “waiting room” type of cavity, where the substrate would plausibly be transferred after the PCD-contraction.…”

Conformational Changes in a Macrolide Antibiotic Binding Protein From Mycobacterium smegmatis Upon ADP Binding

“…The classical organization of ABC transporters requires interaction between two transmembrane domains (TMDs) which usually impart substrate specificity, and two nucleotide-binding domains (NBDs) which couple ATP hydrolysis to drug efflux, such as MacB ( Souabni et al, 2021 ). However, not all NBD containing protein (NP) harbored a predicted TMD partner(s) in the same transcriptional unit or in the vicinity, such as NPs belong to sub-family 3 in Clostridioides difficile ( Pipatthana et al, 2021 ).…”

Functional Overexpression of Membrane Proteins in E. coli: The Good, the Bad, and the Ugly

“…Gold standards are activity assays (in detergent and after reconstitution in lipid bilayers) (e.g. [38][39][40][41] for ABC transporters) and the homogeneous profile in size-exclusion chromatography [34,41]. In addition, high-throughput thermal stability screening can be useful to find detergents that efficiently stabilize membrane proteins [42].…”